Amoeboid motility plays a vital role in such diverse processes as embryonic development, wound healing, cellular immunity, homing to target organs, and tumor metastasis. In most amoeboid cells, locomotion is mediated by a complex, actin-based apparatus. Nematode sperm are an exception. These cells move like other crawling cells by extending a motile pseudopod, but contain a well-ordered, dynamic system of filaments composed of major sperm protein (MSP). Thus, these sperm offer a valuable complement to actin-based systems for investigation of the mechanism of amoeboid movement. This novel motility system will be investigated using sperm of the parasitic nematode, Ascaris suum. In these cells, MSP filaments are organized into well-ordered arrays called fiber complexes, that span the length of the pseudopod and flow rearward at the same rate as the cell crawls forward. Thus, sperm locomotion is closely related to both continuous assembly of MSP filaments at the extreme leading edge of the pseudopod and the arrangement of those filaments into motile fiber complexes. This study will focus on the molecular interactions underlying these key events in motility. The capacity to form crystals and filaments of MSP in vitro will be exploited to examine the molecular structure of the protein and its subunit arrangement in filaments to high resolution using a combination of X-ray diffraction, electron microscopy and computer image processing. Several lines of evidence suggest that the precise control of filament formation in vivo is due to pH-sensitive interaction of MSP with plasma membrane. To test this hypothesis sperm membranes will be isolated and fractionated to identify the components that bind to MSP and analyze quantitatively the effects of membranes and pH on the polymerization process. Proteins that crosslink MSP filaments together to establish the distinctive, three-dimensional architecture of the fiber complexes will be identified and characterized by determining their biochemical properties, their sequence, and their MSP-binding domain. Three complementary strategies, microinjection of labeled analogs of motility proteins to examine their localization and dynamics, disruption of function by incorporation of modified proteins or inhibitory antibodies into intact cells, and reconstitution of function by introduction of motility proteins into pseudopods emptied by hypotonic lysis, will be used to determine the roles of components of the motile apparatus in vivo and deduce the molecular interactions necessary for locomotion. The long-term objective of this proposal is to define the molecular mechanism of sperm locomotion so that comparison of MSP- and actin-based systems can be used to obtain a fuller understanding of the principles of amoeboid motility.